This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. In the 1980's we demonstrated that antibiotics that inhibit bacterial protein synthesis have greater efficacy than cell wall active agents in the treatment of severe group A streptococcal and clostridial myonecrosis. Toxin suppression has become a critically important goal in the treatment of aggressive infections due to group A streptococcus, histotoxic clostridial species and Staphylococcus aureus. The impact of antibiotic-induced toxin upregulation has not been examined in the context of other life-threatening, toxin-mediated infections such as clostridial gas gangrene, streptococcal toxic shock syndrome or Clostridium difficile-associated diarrhea (CDAD). We hypothesize that upregulation of toxic gene expression by Beta-lactam antibiotics occurs in several clinically impotant Gram positive pathogens and is mediated by a common Cell Signaling pathway following engagement of penicillin-binding proteins. In Specific Aim 1 we will elucidate the roles of agr, RNAIII and other toxin regulatory pathways in nafcillin-induced upregulation of exotoxin production in S. aureus. In Specific Aim 2 we will compare the effects of antibiotics associated with triggering CDAD (clindamycin, ampicillin and ciprofloxacin) and those currently used for C. difficile treatment (vancomycin and metronidazole) on production of Toxins A and B by C. difficile. In Specific Aim 3 we will compare the growth phase-dependent transcriptome of group A streptococcus in the presence and absence of subinhibitory concentrations of beta-lactam antibiotics. In specific Aim 4 we will investigate innate immune system recognition of and response to beta-lactam-treated S aureus, group A streptococcus, C perfringens and C. difficile. Understanding the mechanisms responsible for, and the immune consequences of, antibiotic-induced toxin upregulation in these organisms will enable a more rational approach to antibiotic therapy.